Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

Extracellular vesicles from adipose mesenchymal stem cells target inflamed lymph nodes in experimental autoimmune encephalomyelitis.

BACKGROUND AIMS: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. METHODS: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. RESULTS: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. CONCLUSIONS: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.

Tackling TNF-α in autoinflammatory disorders and autoimmune diseases: From conventional to cutting edge in biologics and RNA- based nanomedicines.

Autoinflammatory disorders and autoimmune diseases result from abnormal deviations of innate and adaptive immunity that heterogeneously affect organs and clinical phenotypes. Despite having etiologic and phenotypic differences, these two conditions share the onset of an aberrant inflammatory process. Targeting the main drivers controlling inflammation is useful to treat both autoimmune and autoinflammatory syndromes. TNF-α is a major player in the inflammatory immune response, and anti-TNF-α antibodies have been a revolutionary treatment in many autoimmune disorders. However, production difficulties and high development costs hinder their implementation, and accessibility to their use is still limited. Innovative strategies aimed at overcoming the limitations associated with anti-TNF-α antibodies are being explored, including RNA-based therapies. Here we summarize the central role of TNF-α in immune disorders and how anti-TNF-based immunotherapies changed the therapeutic landscape, albeit with important limitations related to side effects, tolerance, and resistance to therapies. We then outline how nanotechnology has provided the final momentum for the use of nucleic acids in the treatment of autoimmune and autoinflammatory diseases, with a focus on inflammatory bowel diseases (IBDs). The example of IBDs allows the evaluation and discussion of the nucleic acids-based treatments that have been developed, to identify the role that innovative approaches possess in view of the treatment of autoinflammatory disorders and autoimmune diseases.

Immune checkpoint blockade therapy mitigates systemic inflammation and affects cellular FLIP-expressing monocytic myeloid-derived suppressor cells in non-progressor non-small cell lung cancer patients.

Cancer cells favor the generation of myeloid cells with immunosuppressive and inflammatory features, including myeloid-derived suppressor cells (MDSCs), which support tumor progression. The anti-apoptotic molecule, cellular FLICE (FADD-like interleukin-1ß-converting enzyme)-inhibitory protein (c-FLIP), which acts as an important modulator of caspase-8, is required for the development and function of monocytic (M)-MDSCs. Here, we assessed the effect of immune checkpoint inhibitor (ICI) therapy on systemic immunological landscape, including FLIP-expressing MDSCs, in non-small cell lung cancer (NSCLC) patients. Longitudinal changes in peripheral immunological parameters were correlated with patients' outcome. In detail, 34 NSCLC patients were enrolled and classified as progressors (P) or non-progressors (NP), according to the RECIST evaluation. We demonstrated a reduction in pro-inflammatory cytokines such as IL-8, IL-6, and IL-1ß in only NP patients after ICI treatment. Moreover, using t-distributed stochastic neighbor embedding (t-SNE) and cluster analysis, we characterized in NP patients a significant increase in the amount of lymphocytes and a slight contraction of myeloid cells such as neutrophils and monocytes. Despite this moderate ICI-associated alteration in myeloid cells, we identified a distinctive reduction of c-FLIP expression in M-MDSCs from NP patients concurrently with the first clinical evaluation (T1), even though NP and P patients showed the same level of expression at baseline (T0). In agreement with the c-FLIP expression, monocytes isolated from both P and NP patients displayed similar immunosuppressive functions at T0; however, this pro-tumor activity was negatively influenced at T1 in the NP patient cohort exclusively. Hence, ICI therapy can mitigate systemic inflammation and impair MDSC-dependent immunosuppression.

"Open Sesame" to the complexity of pattern recognition receptors of myeloid-derived suppressor cells in cancer.

Pattern recognition receptors are primitive sensors that arouse a preconfigured immune response to broad stimuli, including nonself pathogen-associated and autologous damage-associated molecular pattern molecules. These receptors are mainly expressed by innate myeloid cells, including granulocytes, monocytes, macrophages, and dendritic cells. Recent investigations have revealed new insights into these receptors as key players not only in triggering inflammation processes against pathogen invasion but also in mediating immune suppression in specific pathological states, including cancer. Myeloid-derived suppressor cells are preferentially expanded in many pathological conditions. This heterogeneous cell population includes immunosuppressive myeloid cells that are thought to be associated with poor prognosis and impaired response to immune therapies in various cancers. Identification of pattern recognition receptors and their ligands increases the understanding of immune-activating and immune-suppressive myeloid cell functions and sheds light on myeloid-derived suppressor cell differences from cognate granulocytes and monocytes in healthy conditions. This review summarizes the different expression, ligand recognition, signaling pathways, and cancer relations and identifies Toll-like receptors as potential new targets on myeloid-derived suppressor cells in cancer, which might help us to decipher the instruction codes for reverting suppressive myeloid cells toward an antitumor phenotype.

Nanovesicles from adipose-derived mesenchymal stem cells inhibit T lymphocyte trafficking and ameliorate chronic experimental autoimmune encephalomyelitis.

Cell based-therapies represent promising strategies for the treatment of neurological diseases. We have previously shown that adipose stem cells (ASC) ameliorate chronic experimental autoimmune encephalomyelitis (EAE). Recent evidence indicates that most ASC paracrine effects are mediated by extracellular vesicles, i.e. micro- and nanovesicles (MVs and NVs). We show that preventive intravenous administration of NVs isolated from ASC (ASC-NVs) before disease onset significantly reduces the severity of EAE and decreases spinal cord inflammation and demyelination, whereas therapeutic treatment with ASC-NVs does not ameliorate established EAE. This treatment marginally inhibits antigen-specific T cell activation, while reducing microglial activation and demyelination in the spinal cord. Importantly, ASC-NVs inhibited integrin-dependent adhesion of encephalitogenic T cells in vitro, with no effect on adhesion molecule expression. In addition, intravital microscopy showed that encephalitogenic T cells treated with ASC NVs display a significantly reduced rolling and firm adhesion in inflamed spinal cord vessels compared to untreated cells. Our results show that ASC-NVs ameliorate EAE pathogenesis mainly by inhibiting T cell extravasation in the inflamed CNS, suggesting that NVs may represent a novel therapeutic approach in neuro-inflammatory diseases, enabling the safe administration of ASC effector factors.

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency.

RATIONALE: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. OBJECTIVES: We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. METHODS: We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. MEASUREMENTS AND MAIN RESULTS: We found that chemoattractant-induced activation of ß1 and ß2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of ß1 and ß2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. CONCLUSIONS: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type-selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis.